3. Fungal and Bacterial Resistance
Plant pathogens reduce crop yields by adversely affecting plant growth and development. It has been estimated that diseases globally reduce crop yields by 20–40%
[49]. In the past decade, there has been a surge in the use of genome editing technologies in generating crops that are resistant to a wide range of pathogens. Several studies of targeted mutagenesis in crop plants, including deletions, insertions, and replacement of DNA of various sizes at targeted sites, have proven to be a promising approach to improve plant resistance to pathogens.
Developing plant resistance by modifying host S genes such as those belonging to the
mlo (Mildew Resistant Locus O) gene family has been effective in apple, tomato, barley, and wheat
[50]. However, a common disadvantage of S gene mutation is the concomitant negative impact on plant growth and productivity
[51][52]. Therefore, a CRISPR/Cas9-induced targeted deletion in the
MLO-B1 locus resulted in a mutant wheat variety (Tamlo-R32) that thrived better and maintained growth and yields while conserving resistance to powdery mildew
[53]. In another study conducted by Peng et al.
[54] on citrus plants, CRISPR/Cas9-targeted modification of the S gene
CsLOB1 was performed to enhance resistance to citrus canker. Some recent experiments on rice reported the use of CRISPR/Cas9 technology to induce mutagenesis in the promoter region of bacterial blight S genes,
OsSWEET14 and
OsSWEET11 [55]. Antony et al.
[56] developed blight-resistant rice plants with an
OsSWEET14 TDNA insertion mutant but when compared to wild-type plants the mutants had smaller seeds. In contrast, resistance against rice blight with no growth defects was detected in the TALE-edited
OsSWEET14 gene in super basmati rice
[57]. These results suggest that engineering S genes through genome editing technology is a potential strategy to enhance rice resistance to blight caused by
Xanthomonas oryzae pv. Oryzae. Thus, with rice production threatened by bacterial blight causing major crop losses, rapid and durable methods are desperately needed.
In addition to the knockout of host genes to improve disease resistance, endophyte-derived genes can serve as additional routes for improvement of wheat traits. In a recent study by Wang et al.
[58], the authors identified a Fusarium resistance gene (
Fhb7)
, which was shown to be horizontally transferred from an endophytic fungus and conferred resistance to Fusarium head blight (FHB), a significant fungal disease of wheat. The researchers demonstrated that introgression of
Fhb7 into the genome of many commercial wheat cultivars conferred tolerance to FHB without negatively affecting growth yields, suggesting that
Fhb7 is a potential candidate for engineering blight resistance in elite wheat varieties. Similarly, in tomato plants, CRISPR/Cas 9-mediated knockout of the
DMR6 gene enhanced resistance to bacterial pathogens including
P. syringae, P. capsici, and
Xanthomonas spp., with no adverse effects on plant development and growth. As more targets are identified, it is expected that there will be many more successful studies on durable and broad-spectrum disease resistance using the CRISPR/Cas9 approach in a wide range of crops.
The effect of overexpression of plant proteins such as ribosome-inactivating proteins (RIPs) on performance of fungal and bacterial pathogens has been investigated
[59]. Some interesting examples, such as overexpression of the
PhRIP gene in transgenic potato, protected the plants against damage from
Botrytis cinerea and
Rhizoctonia solani, whereas expressing the RIP
alpha-MMC gene improved resistance to rice blast fungus in rice
[60][61]. Other proteins, including PFLP (plant ferredoxin-like protein) and HRAP (hypersensitive response-assisting proteins), are effective against multiple bacterial pathogens when they are overexpressed in rice, banana, and other species
[62][63]. Recently, overexpression of
PFLP and
HRAP genes in greenhouse and field-grown bananas indicated that both genes are effective against bacterial wilt caused by
Xanthomonas spp.
[64]. In an earlier study, a combination of both genes did not provide any additional benefits in banana, yet the authors speculate that bananas expressing both genes may be more durable
[65]. Furthermore, the disease-resistance trait was passed on to the next generation of transgenic lines. Additionally, the authors noted no difference in the agronomic traits, including yield and flowering of field-grown symptom-free bananas. These examples are helpful in revealing the biological function of plant proteins that can be beneficial when engineering crop tolerance to pathogen attacks.
Harpins may act in extracellular spaces in plant tissue, facilitating recognition by the plant
[66]. Harpins are effective against multiple pathogens when overexpressed in tobacco, rice, canola, and cotton
[67][68][69][70][71]. The effect of overexpression of harpin
xooc encoding the
hrf2 gene on the performance of an oomycete pathogen was investigated by Niu et al.
[72]. The results demonstrate that transgenic soybeans expressing the
hrf2 gene showed enhanced resistance to
Phytophthora sojae. This study provides a valuable insight toward the functional role of the
hrf2 gene in plant defense against
P. sojae, opening new avenues for understanding other important pathogens as well as subsequently engineering broad-spectrum disease resistance in soybean.
The ability of plants to utilize diverse classes of immune receptors to perceive the presence of pathogenic microbes makes possible the transferring and engineering of these receptors to improve recognition capacities
[73]. Recently, Ercoli et al.
[74] conducted studies to show how the XA21 receptor in rice recognizes and resists infection caused by
Xanthomonas oryzae. Other immune receptors have been targeted for modification in potato, apple, and rice. A late blight-resistant potato with a NOD-like receptor (NLR) introduced from a wild relative is currently on the market in the UK
[12]. In apples, modification of the
HcrVf2 gene encoding such a receptor conferred resistance against the devastating fungal scab
Venturia inaequalis [75]. Xu and colleagues
[76] showed that rice plants constantly expressing an immune regulator gene, NPR1 (non-expressor of pathogenesis-related genes 1), conferred resistance to bacterial blight but displayed growth defects. However, when the authors controlled NPR1 expression, it resulted in increased accumulation of NPR1 upon pathogen infection, enhancing resistance to bacterial blight without negatively affecting plant growth and grain yield. Although this method enables researchers to obtain plants with strong immunity, durability can be challenging because pathogens are evolving rapidly.
An important strategy in the fight against Verticillium wilt (VW), caused by fungi belonging to the genus
Verticillium, is to silence genes essential for spore production, hyphal development, and pathogenicity. RNAi-mediated silencing of the
VdRGS1 gene has been achieved in cotton, resulting in transgenic plants with enhanced resistances to VW. With the help of RNAi technology, Govindrajulu et al.
[77] showed that the transgenic lettuce containing a modified construct of highly abundant message #34 (
HAM34) and cellulose synthase (
CES1) genes showed resistance against a biotrophic pathogen that causes downy mildew of lettuce. Similarly, a
PsFUZ7 RNAi construct expressed in transgenic wheat conferred strong resistance to wheat stripe rust. Additionally, knocking down the transcription factor gene
OsERF922 (ethylene responsive factor) using RNAi results in increased resistance against the pathogen
Magnoporthe oryzae [78]. The improvement of rice blast resistance via CRISPR/Cas9, which targeted knockdown of the
OsERF922 gene in a japonica rice variety cultivated in northern China, was reported by Wang et al.
[79]. This modification led to no detrimental effects on rice growth and development. Overall, RNAi appears to be a promising approach to control the detrimental effects of many fungi and oomycetes.
4. Increasing Plant Yield
One of the main objectives of growing plants, whether the plants are transgenic or non-transgenic, is to increase the yield of the target plant. Moreover, plant yield is important whether the plant is intended to be used as a food or as the source of a cloned protein. In addition, increasing crop yields can decrease the amount of land that is required for agricultural production
[80].
In recent years, scientists have developed several unique schemes intended to increase the yield of various plants. For example, one group of scientists developed an approach that they called ‘speed breeding’ that is applicable to essentially all crops
[81]. Speed breeding entails extending the plant’s photoperiod, controlling the plant’s growth temperature, and selection of fast-growing seeds. Of course, to more rapidly grow plants, controlling their growth environment requires the extensive use of greenhouses. In this environment, the lighting may be supplemented with artificial electric lamps
[82], the photoperiod may be extended
[83], and the wavelength of the lighting may be altered
[84]. Following these protocols, researchers have reduced the growth cycle of many different plants to an average of half of what it was previously, thereby enabling the controlled growth of additional generations of the same plant
[81].
Scientists have developed several different schemes in an effort to increase plant crop yields. For example, traditional varieties of wheat and rice allocate a significant fraction of their resources to producing vegetative tissues rather than grain or reproductive tissues
[85]. However, semi-dwarf varieties previously developed by conventional breeding during the so-called green revolution
[86] allocate a greater portion of their resources to grain rather than to vegetative (leaf) tissues. By genetic modulation of the levels of the phytohormones gibberellin and brassinosteroid, it should be possible to produce dwarf plant strains that allocate even more resources to grain instead of vegetative tissues, and thereby further increase the grain yield.
In another study, researchers observed that they were able to genetically modify tomato plants to increase their harvest index, which is the ratio of fruit yield to total plant biomass
[87]. This was accomplished by introducing a chloroplast-targeted cyanobacterial flavodoxin gene into tomato plants. In this case, the transgenic plants were generally smaller than the wild-type plants with a higher number of tomato fruits per plant. Moreover, the overall yield of tomato fruit could be augmented by increasing the density of plants in the field (i.e., by planting the transgenic plants close together). In a separate study, scientists increased the expression of the maize (corn) MADS-box transcription factor gene
zmm28 by placing this gene under the control of a moderate-level constitutive maize promoter
[88]. In field trials, the transgenic plants with increased expression of the
zmm28 gene showed an increase in carbon assimilation, nitrogen utilization, and plant growth, all leading to an increase in grain yield relative to the cultivar of wild-type maize used in this study. Methylation of the N
6 position of adenosine residues in RNA molecules is common in plants (as well as in other higher eukaryotes), and this modification is believed to regulate RNA processing and metabolism
[89]. In a recent experiment, scientists introduced and expressed the human demethylase FTO gene into rice and potato plants
[90]. Expression of this transgene resulted in an exceptionally large increase in rice grain yields in plants that were grown in the greenhouse and a smaller, although still highly significant, increase in rice and potato yield and biomass when plants were grown in the field. In this experiment, expression of the transgene caused an increase of root meristem proliferation and tiller bud formation as well as overall plant photosynthetic efficiency and drought tolerance, suggesting that modulating RNA methylation is an effective way to improve plant yield. Scientists who generated transgenic rice by overexpressing the rice gene encoding a plasma membrane H
+-ATPase 1 gene (i.e.,
OSA1) found that the resultant transgenic plants significantly improved their utilization of nitrogen and carbon resources compared to wild-type
[91]. As a result of this manipulation, the transgenic plants showed a large increase in grain yield.
In addition to introducing exogenous genes to increase plant yield, some researchers have used the CRISPR/Cas9 system to modify some of the existing plant genes (i.e., this technique enables scientists to directly alter specific plant genes) to achieve the same ends as genetic transformation
[92]. In one instance, in the T2 generation following the genomic modification, three of the four rice plant mutants that were created yielded an increased number of rice grains and had a larger grain size. Also using the CRISPR/Cas9 system, the alteration of multiple genes in a single tomato cultivar worked together to affect the yield of rice. In this case, the CRISPR/Cas9 system was used to edit six independent loci that were important for controlling the yield and productivity in a wild tomato (
Solanum pimpinellifolium) crop line
[93]. When the engineered tomato plant was compared to the wild-type plant, it was observed to have a three-fold increase in fruit size and a ten-fold increase in fruit number. In addition to these examples, researchers have used the CRISPR/Cas9 system to modify an already shortened version of canola (by conventional breeding) so that the plant has more branches, resulting in the formation of more flowers and pods. This was achieved by knocking out genes for receptors that perceive the hormone strigolactone
[94]. Another group of researchers used the CRISPR/Cas9 system to knock out the rice
OsPDCD5 gene, which is involved in programmed cell death
[95]. Mutating this gene decreased auxin synthesis by the modified plant in addition to lowering gibberellin and cytokinin synthesis and signaling pathways. Moreover, rice that contained mutations in the
OsPDCD5 gene had an increased yield of rice grains.
From the above cursory exploration of some strategies that have been employed to increase plant fruit or grain yield, it is clear that plant yield is controlled by a relatively large number of different genes. The expression of some of these genes may be increased while the expression of others is decreased with the same ultimate result, i.e., the yield is increased. Moreover, in some instances, the addition of foreign genes may also result in an increase in yield. While most of these genetic manipulations have yet to be tested and proven in the field, there is every reason to expect that the approaches described here will eventually lead to plants with much greater fruit, seed, and grain yield than is currently available from wild-type plants, including plants that have been manipulated by traditional breeding techniques.